核桃NBS类抗病基因类似物的序列特征及其与炭疽病的抗性

doi:10.3864/j.issn.0578-1752.2014.02.014

摘要/Abstract

摘要： 【目的】利用同源序列法分离核桃的NBS（Nucleotide binding site）类抗病基因类似物，为核桃抗炭疽病分子辅助育种及抗病基因的克隆提供基础。【方法】以35个核桃优系为试材，利用接种法鉴定供试材料对炭疽病的抗性；根据已知植物抗病基因的保守结构域P-loop和GLPL设计简并引物，以核桃优系的基因组DNA为模板，通过PCR扩增NBS类抗病基因同源序列片段，并分析所得NBS类抗病基因类似物与核桃优系炭疽病抗性的关系；利用BLASTN/X程序对所得NBS序列在GenBank数据库中进行同源性搜索；利用MEGA 5.2及DNAman 7.0等软件对其进行序列相似性分析和系统进化研究。【结果】35个供试核桃优系中，有20个优系为抗炭疽病类型（R），其相对抗性指数在0.63—0.82；有5个优系为中抗类型（M），相对抗性指数在0.27—0.56；有10个为感病类型（S），相对抗性指数在0.00—0.21。PCR结果显示，从20个抗炭疽病优系的基因组扩增得到20条NBS类抗病基因类似序列；而在其它15个优系（5个中抗优系和10个感病优系）中未扩增到条带，表明核桃NBS序列与炭疽病抗性相关联。BLASTN显示，所得NBS序列在核苷酸水平与GenBank中已知的核桃NBS序列(jrRGAPGs)存在89%—100%同源性，与其它物种NBS序列的同源性在69%以上；BLASTX显示，所得NBS序列与已知核桃NBS抗病蛋白同源性为77%—99%，与其它物种的NBS抗病蛋白同源性在49%—66%。氨基酸序列多重比对分析表明，所得核桃NBS序列均包含有抗病基因所具有的典型功能域，如P-loop、kinase-2、kinase-3和GLPL等，在典型功能域的核苷酸多态性（Pi）明显低于非保守区，有较高保守性。系统发育分析表明，在核苷酸水平上可将所得核桃优系的NBS序列区分为7类，在氨基酸水平上可分TIR和non-TIR两大类7个亚类；所得核桃NBS序列非同义替换率(dN)和同义替换率(dS)的比值dN/dS在0.00—0.95，为纯化选择。氨基酸序列相似性表明核桃优系不同NBS亚类间的相似度为28.3%—63.5%，与已知抗病基因相应区域的氨基酸相似性为22.0%—48.5%。【结论】核桃NBS类抗病基因类似物与炭疽病抗性相关联，NBS序列与其他物种抗病基因有较高同源性，包含抗病基因保守的功能域，在进化上为纯化选择。

关键词: 核桃 , 炭疽病 , 抗病基因类似物 , 核苷酸多态性 , 系统发育分析

Abstract: 【Objective】Isolating NBS-type （Nucleotide binding site） resistance gene analogs (RGAs) from walnut (Juglans regia L.) using homology-based method would provide a foundation for molecular-assisted selection and for cloning R gene during walnut breeding. 【Method】Thirty-five walnut superiors were used as plant materials in the study and their resistance to anthracnose was identified with inoculation. NBS-type RGAs were isolated by a PCR strategy using degenerate primers specific to P-loop and GLPL, conserved motifs of NBS domain in plant R gene. The relationship between NBSs and walnut anthracnose resistance was analyzed. Sequence identification of obtained sequences was performed against known RGAs deposited in GenBank using BLASTN/X algorithms. Similar and phylogenetic analysis were elaborated using MEGA 5.2 and DNAman 7.0 software.【Result】Out of 35 tested walnut superiors, 20 superiors were identified as resistant (R) with a relative resistance index (RRI) ranging from 0.63 to 0.82; 5 of them were medium resistant (M), whose RRI ranged 0.27-0.56; the rest 10 superiors as susceptible (S) with the RRI ranging 0.00-0.21. NBSs were amplified only in 20 resistant superiors and no bands were found in the remaining 15 superiors (5 medium resistant and 10 susceptible), indicating that the NBS-RGAs were associated with the resistance to walnut anthracnose (Colletotrichum gloeosporioides). BLASTN showed the obtained NBSs shared high similarities to cloned jrRGAPGs with a identity ranging 89%-100%; and they shared than 69% homology to other species from GenBank; BLASTX revealed 77%-99% and 49%-66% similarity to the NBS proteins from jrRGAPGs and other species, respectively. Multiple alignment analysis revealed that these NBS-type RGAs contained some well-characteristics motifs of NBS genes, including P-loop, kinase-2, kinase-3 and GLPL. The nucleotide polymorphism and diversity (Pi) were highly conserved at each motif than non-conservative fragments, indicating their conservative structures. Phylogenetic analysis revealed that the NBSs were clustered into seven subgroups at nucleotide level. They were grouped into two clades (TIR and non-TIR) and were subdivided into 7 groups based on their amino acid sequence similarity. Ratio of non-synonymous to synonymous nucleotide substitution (dN/dS) among NBS-RGAs varied from 0.00 to 0.95 (lower than one) for different classes, suggesting a purifying selection. Similarity percentages of deduced amino acid among these 7 NBS subgroups ranged from 28.3% to 63.5% with identifies to R genes ranging from 22.0% to 48.5%. 【Conclusion】 NBS-type RGAs isolated in the study were associated with anthracnose resistance, they were highly similar to cloned R genes and contained some conserved motifs. Walnut NBS-type RGAs undergo a purifying selection.

Key words: walnut (Juglans regia L.) , walnut anthracnose (Colletotrichum gloeosporioides) , resistance gene analogs (RGA) , nucleotide polymorphism and diversity (Pi) , phylogenetic analysis

安海山1, 杨克强1, 2. 核桃NBS类抗病基因类似物的序列特征及其与炭疽病的抗性[J]. 中国农业科学, 2014, 47(2): 344-356.

AN Hai-Shan-1, YANG Ke-Qiang-1, 2 . Sequence Analysis of NBS-Type RGAs and Their Relationship with Anthracnose Resistance in Walnut[J]. Scientia Agricultura Sinica, 2014, 47(2): 344-356.

0
/ / 推荐

导出引用管理器 EndNote|Reference Manager|ProCite|BibTeX|RefWorks

链接本文: https://www.chinaagrisci.com/CN/10.3864/j.issn.0578-1752.2014.02.014

https://www.chinaagrisci.com/CN/Y2014/V47/I2/344

参考文献

[1]李敏, 刘媛, 孙翠, 孟亚楠, 杨克强, 侯立群, 王钧毅. 核桃营养价值研究进展. 中国粮油学报, 2009, 24(6): 166-170.

Li M, Liu Y, Sun C, Meng Y N, Yang K Q, Hou L Q, Wang J Y. Research advance about nutrients and medicinal value of walnut. Journal of the Chinese Cereals and Oils Association, 2009, 24(6): 166-170. (in Chinese)

[2]曲文文, 杨克强, 刘会香, 王钧毅. 山东省核桃主要病害及其综合防治. 植物保护, 2011, 7(2): 136-140.

Qu W W, Yang K Q, Liu H X, Wang J Y. Main disease of walnut (Juglans regia L.) and the integrate management in Shandong province. Plant Protection, 2011, 7(2): 136-140. (in Chinese)

[3]刘霞, 杨克强, 朱玉凤, 尹燕飞. 8种杀菌剂对核桃炭疽病病原菌胶孢炭疽菌的室内毒力. 农药学学报, 2013, 15(4): 412-410.

Liu X, Yang K Q, Zhu Y F, Yin Y F. The laboratory toxicity of eight fungicides to Colletotrichum gloeosporioides caused walnut anthracnose. Chinese Journal of Pesticide Science, 2013, 15(4): 412-410. (in Chinese)

[4]阙友雄, 许莉萍, 林剑伟, 张木清, 陈如凯. 斑茅NBS-LRR类抗病基因同源序列的克隆与分析. 热带作物学报, 2009, 30(2): 192-197.

Que Y X, Xu L P, Lin J W, Zhang M Q, Chen R K. Cloning and analysis of NBS-LRR type disease resistance gene analogs in Erianthus arundinaceum. Chinese Journal of Tropical Crops, 2009, 30(2): 192-197. (in Chinese)

[5]Kanazin V, Marek L F, Shoemaker R C. Resistance gene analogs are conserved and clustered in soybean. Proceedings of the National Academy of Sciences of the USA, 1996, 93: 11746-11750.

[6]Meyers B C, Dickerman A W, Michelmore R W, Sivaramakrishnan S, Sobral B W, Young N D. Plant disease resistance genes encode members of an ancient and diverse protein family within the nucleotide binding superfamily. The Plant Journal, 1999, 20(3): 317-332.

[7]阙友雄, 许莉萍, 林剑伟, 陈如凯. 甘蔗NBS-LRR类抗病基因同源序列的分离与鉴定. 作物学报, 2009, 35(4): 631-639.

Que Y X, Xu L P, Lin J W, Chen R K. Isolation and characterization of NBS-LRR resistance analogs from sugarcane. Acta Agronomica Sinica, 2009, 35(4): 631-639. (in Chinese)

[8]丁国华, 池春玉, 周秀艳, 秦智伟. 黄瓜抗病基因类似序列(RGA)的同源性分析和Southern鉴定. 园艺学报, 2007, 34(2): 355-360.

Ding G H, Chi C Y, Zhou X Y, Qin Z W. Southern identification and homology analysis of the resistance gene analogs in Cucumis sativus L.. Acta Horticulturae Sinica, 2007, 34(2): 355-360. (in Chinese)

[9]Gururani M A, Venkatesh J, Upadhyaya C P, Nookaraju A, Pandey S K, Park S W. Plant disease resistance genes: Current status and future directions. Physiological and Molecular Plant Pathology, 2012, 78: 51-65.

[10]Shi A, Kantartzi S K, Mmbaga M, Chen P. Isolation of resistance gene analogues from flowering Dogwood (Cornus florida L.). Journal of Phytopathology, 2008, 156: 742-746.

[11]Xu Q, Wen X, Deng X. Isolation of TIR and nonTIR NBS-LRR resistance gene analogues and identification of molecular markers linked to a powdery mildew resistance locus in chestnut rose (Rosa roxburghii Tratt). Theoretical and Applied Genetics, 2005, 111: 819-830.

[12]Yu Y G, Buss G R, Maroof M A. Isolation of a superfamily of candidate disease resistance genes in soybean based on a conserved nucleotide-binding site. Proceedings of the National Academy of Sciences of the USA, 1996, 93: 11751-11756.

[13]Joshi R K, Mohanty S, Subudhi E, Nayak. Isolation and characterization of NBS-LRR resistance gene candidates in turmeric (Curcuma longa cv. surama). Genetics Molecular Research, 2010, 9(3): 1796-1806.

[14]Shevelukha V S, Kuklev M Y, Karlov G I. Cloning and characterization of NBS-LRR class resistance gene analogs sequences in Sunflower. Acta Agriculturae Serbica, 2003, 8(15): 3-9.

[15]Lalli D A, Decroocq V, Blenda A V, Schurdi-Levraud V, Garay L, Damsteegt O V, Reighard G L, Abbortt A G. Identification and mapping of resistance gene analogs (RGAs) in Prunus: A resistance map for Prunus. Theoretical and Applied Genetics, 2005, 111: 1504-1513.

[16]李宁, 黄茜, 刘燕, 赵丹, 刘艳, 黄占景, 张增艳. 小麦抗病基因类似序列BRG1的分离与功能分析. 作物学报, 2011, 37(6): 998-1004.

Li N, Huang X, Liu Y, Zhao D, Liu Y, Huang Z J, Zhang Z Y. Identification and functional analysis of a wheat resistance analogous gene BRG1. Acta Agronomica Sinica, 2011, 37(6): 998-1004. (in Chinese)

[17]Fahrentrapp J, Broggini G A L, Kellerhals M, Peil A, Richter K, Zini E, Gessler C. A candidate gene for fire blight resistance in Malus × robusta 5 is coding for a CC-NBS-LRR. Tree Genetics and Genomes, 2013, 9: 237-251.

[18]Li J, Ding J, Zhang W, Zhang Y, Tang P, Chen J Q, Tian D, Yang S. Unique evolutionary pattern of numbers of gramineous NBS-LRR genes. Molecular Genetics and Genomics, 2010, 283: 427-438.

[19]Pollegioni P, Van der Linden G, Belisario A, Gras M, Anselmi N, Olimpieri I, Luongo L, Santini A, Turco E, Mugnozza G S, Malvolti M E. Mechanisms governing the responses to anthracnose pathogen in Juglans spp. Journal of Biotechnology, 2012, 159: 251-264.

[20]刘庆忠, 张力思. 核桃种质资源描述规范和数据标准. 北京: 中国农业出版社, 2007.

Liu Q Z, Zhang L S. Descriptors and Data Standard for Walnut (Juglans regia L.). Beijing: China Agriculture Press, 2007. (in Chinese)

[21]何建群, 陈贵荟, 李靖军, 王家兰, 杨万春, 杨芸, 周进行. 亚麻品种白粉病田间抗病性分析. 中国麻业科学, 2007, 29(3): 141-144.

He J Q, Chen G H, Li J J, Wang J L, Yang W C, Yang Y, Zhou J X. The analysis of resistance of flax to powdery mildew (Olidium lini skoric) in fild. Plant Fiber Sciences in China, 2007, 29(3): 141-144. (in Chinese)

[22]杨克强, 马明, 孙彩玲, 孙明高, 郭起荣. 核桃早实基因的RAPD标记及其序列分析研究. 中国农业科学, 2007, 40(9): 2021-2027.

Yang K Q, Ma M, Sun C L, Sun M G, Guo Q R. RAPD marker linked to precocious gene of walnut (Juglans regia L.) and its sequence analysis. Scientia Agricultura Sincia, 2007, 40(9): 2021-2027. ( in Chinese)

[23]Seehalak W, Moonsom S, Metheenukul P, Tantasawat P. Isolation of resistance gene analogs from grapevine resistant and susceptible to downy mildew and anthracnose. Scientia Horticulturae, 2011, 128: 357-363.

[24]李春来, 张怀渝. 植物抗病基因同源序列(RGA)研究进展. 分子植物育种, 2004, 2(6): 853-860.

Li C L, Zhang H Y. Research advance of resistance gene analogs in plant. Molecular Plant Breeding, 2004, 2(6): 853-860. (in Chinese)

[25]孔凡晶, 马有志, 陈孝, 辛志勇. 簇毛麦端体6VS的抗病同源序列的克隆及分析. 中国农业科学, 2003, 36(10): 1223-1227.

Kong F J, Ma Y Z, Chen X, Xin Z Y. Cloning and characterization of a family of disease resistance gene analogs from 6VS of Haynaldia villosa. Scientia Agricultura Sincia, 2003, 36(10): 1223-1227. (in Chinese)

[26]张丽英, 陈儒刚, 张俊红, 欧阳波, 肖景华, 李汉霞, 叶志彪. 辣椒抗病基因同源序列的克隆与分析. 中国农业科学, 2008, 41(1): 169-175.

Zhang L Y, Chen R G, Zhang J H, OuYang B, Xiao J H, Li H X, Ye Z B. Cloning and analysis of resistance gene analogs from pepper (Capsicum annuum L.). Scientia Agricultura Sincia, 2008, 41(1): 169-175. (in Chinese)

[27]He L M, Du C G, Covaleda L, Xu Z Y, Robinson A F, Yu J Z, Kohel R J, Zhang H B. Cloning, characterization and evolution of the NBS-LRR-encoding resistance gene analogue family in polyploidy cotton (Gossypium hirsutum L.). Molecular Plant-Microbe Interactions, 2004, 17: 1234-1241.

[28]陈观水, 周以飞, 林生, 潘大仁. 甘薯NBS类抗病基因类似物的分离与序列分析. 热带亚热带植物学报, 2006, 14(5): 359-365.

Chen G S, Zhou Y F, Lin S, Pan D R. Isolation and sequence analysis of NBS-type resistance gene analogous in sweet potato(Ipomoea batatas(L.) Lam.). Journal of Tropical and Subtropical Botany, 2006, 14(5): 359-365. (in Chinese)

[29]Palomino C, Satovic Z, Cubero J I, Torres A M. Identification and characterization of NBS-LRR class resistance gene analogs in fafa bean (Vicia fafa L.) and chickpea (Cicer arietinum L.). Genome, 2006, 49: 1227-1237.

[30]Zhang Q, Zhang Z Y, Lin S Z, Zheng H Q, Lin Y Z, An X M, Li Y, Li H X. Characterization of resistance gene analogs with a nucleotide binding site isolated from a triploid white poplar. Plant Biology, 2008, 10: 310-312.

[31]Pan Q, Wendel J, Fluhr R. Divergent evolution of plant NBS-LRR resistance gene homologues in dicot and cereal genomes. Journal of Molecular Evolution, 2000, 50: 203-213.

[32]Rocha E P C, Smith J M, Hurst L D, Holden M T G, Copper J E, Smith N H, Feil E J. Comparison of dS/dN are time dependent for closely related bacterial genomes. Journal of Theoretical Biology, 2006, 239: 226-235.

[33]Mustonen V, Lassig M. Adaptation to fluctuating selection in Drosophila. Proceedings of the National Academy of Sciences of the USA, 2007, 104: 2277-2282.

[34]Cannon S B, Zhu H, Baumgarten A M, Spangler R, May G, Cook D R, Young N D. Diversity, distribution and ancient taxonomic relationships within the TIR and non-TIR NBS-LRR resistance gene subfamlies. Journal of Molecular Evolution, 2002, 54: 548-562.

[35]Reddy B L, Reddy D S, Narsu M L, Sivaramakrishnan S. Characterization of disease resistance gene homologues isolated from finger millet (Eleusine coracana L. Gaertn). Molecular Breeding, 2011, 27: 315-328.

[36]Mondragon-Palomino M, Meyers B C, Michelmore R W, Gaut B S. Patterns of positive selection in the complete NBS-LRR gene family of Arabidopsis thaliana. Genome Research, 2002, 12: 1305-1315.

[37]Mondragon-Palomino M, Gaut B S. Gene conversion and the evolution of three leucine-rich repeat gene families in Arabidopsis thaliana. Molecular Biology and Evolution, 2005, 22: 2444-2456.

[38]Cana E F, Geffroy V, Macadre C, Creusot F, Imbert-Bollore P, Sevignac M, Langin T. Characterization of expressed NBS-LRR resistance gene candidates from common bean. Theoretical and Applied Genetics, 2003, 106: 251-261.